Mammalian skeletal muscle normally undergoes a reparative process after traumatic injury. The process of skeletal muscle repair is actually a series of discrete overlapping events, which can be segregated into trauma, tissue degeneration, inflammation, phagocytosis, angiogenesis, stem cell activation, migration of the stem cells to the site of injury, proliferation of undifferentiated stem cells, reinnervation, differentiation of the stem cells, and remodelling of the tissue.
The early restored muscle tissues approximate embryonic-like myotubes containing centrally-located nuclei and lie adjacent to mature myofibers containing peripherally-located nuclei. Unfortunately, restoration of physiological function is compromised due to the increased proliferative nature of the surrounding connective tissues, eventually forming non-functional scar tissue.
To circumvent the decreased function due to scar tissue formation, Carlson and Faulkner, Med. Sci. Sports Exerc. 15:187-198 (1983); Faulkner and Carlson, Fed. Proc. 45:1454 (1986); and Donovan and Faulkner, J. Appl. Physiol. 62:2507-2511 (1987) severed existing vascular and nervous tissue connections prior to grafting homologous and autologous muscles. This procedure apparently inhibited myogenic differentiation of cultured cells and Faulkner and colleagues reported some restoration of function using it.
Research in other areas has indicated that various factors such as platelet derived growth factor (PDGF), chicken muscle growth factor (CMGF), epidermal growth factor (EGF), sciatic nerve extract, insulin, and somatomedins stimulate a mitogenic or proliferative response in cultured muscle cells. This response should be contrasted with a myogenic response which does not induce myogenic lineage commitment of uncommitted stem cells, but instead induces the lineage commitment of the stem cells.
Three growth factors, insulin and insulin-like growth factors, namely insulin-like growth factor-I (IGF-I), also called somatomedin-C, insulin-like growth factor-II (IGF-II), also called myogenic stimulating activity, have been shown to be potent stimulators of skeletal muscle cell growth and differentiation in cultured myosatellite cells and myogenic lineage-committed stem cells by Ewton and Florini, Dev. Biol. 83:31-39 (1981); Florini et al., J. Biol. Chem. 261:16509-16515 (1986); Florini et al., Am. J. Physiol. 250:C771-778 (1986); Sejersen et al., Proc. Natl. Acad. Sci. 83:6844-6848 (1986); Ewton et al., Fed. Proc. 45:1454-1455 (1987).
Several in vivo studies have employed basic-fibroblast growth factor (b-FGF), transforming growth factor beta (TGF-.beta.), and epidermal growth factor (EGF) to stimulate internal wound healing. Buckley et al., Proc. Natl. Acad. Sci. 82:7340-7344 (1985); Grotendorst et al., J. Clin. Invest. 76:2323-2329 (1985); Franklin et al., J. Lab Clin. Med. 108:103-108 (1986); Roberts et al., Proc. Natl. Acad. Sci. 83:4167-4171 (1986); Mustoe et al., Science 1987; Sprugel et al., Am. J. Path. 129:601-613 (1987); and Davidson et al., Prog. Clin. Biol. Res. (1988) noted that administration of b-FGF, TGF-.beta.B, and EGF appeared to promote proliferation of connective tissue elements to form scar tissue and thus aid in wound healing of mammalian skeletal muscle.
In vitro studies have demonstrated the influence of other growth factors on the resultant phenotypic expression in myogenic cultures. For example, Hauschka and co-workers have reported that acidic-fibroblast growth factor (a-FGF) and basic-fibroblast growth factor (b-FGF) play a dual role in stimulating myoblast proliferation while directly repressing terminal differentiation, as described by Linkhart et al., Dev. Biol. 86:19-30 (1981), the chapter of Linkhart et al. entitled "Control of mouse myoblast commitment to differentiation by mitogens." In: Molecular and Cellular Control of Muscle Development. M. L. Pearson and H. F. Epstein, eds., Cold Spring Harbor Laboratory, New York, p. 377-382 (1982); Lim and Hauschka, J. Cell Biol. 98:739-747 (1984); and Olwin and Hauschka, Biochemistry 25:3487-3492 (1986).
Unfortunately, the administration of growth factors that inhibit terminal myogenic differentiation, promote myoblast proliferation, and promote fibroblast proliferation and differentiation as a method to promote muscle repair appears to cause an increase in connective tissue scar formation. In muscle, increased scar formation creates decreased physiological function. A decrease in connective tissue scar formation with a compensatory increase in skeletal muscle mass plus revascularization and reinnervation of the tissues is necessary for the restoration of physiological function.
Implantation of demineralized bone into ectopic sites (i.e., intramuscular pouch, subcutaneous pouch) result in the induction of cartilage and bone within these tissues, as reported by Urist, Science 150:194-199 (1965); Urist et al., Clin. Orthopaed 64:194-220 (1969); and Reddi and Huggins, Proc. Nat. Acad. Sci. USA 69:1601-1605 (1972). Young et al, Anat. Rec. 223:231-241 (1989), demonstrated the existence of stem cells, arising from the connective tissues associated with muscle, cartilage, and bone, that contributed to the blastemal population during epimorphic limb regeneration in the adult terrestrial salamander. Combined, this work suggest the potential for the presence of quiescent uncommitted stem cells or quiescent lineage-committed stem cells located within or nearby these tissue matrices that upon the appropriate stimulus would form differentiated tissues. The stimulus or stimuli required for differentiation is not known.
A number of delivery systems have been proposed for administration of various drugs and other biologically active compounds. The most promising for practical reasons are the polymer based systems, as described, for example, for general drug delivery using the bioerodible polyanhydrides, in U.S. Pat. No. 4,906,474 to Langer, et al., U.S. Pat. No. 4,888,176 to Langer, et al., and U.S. Pat. No. 4,757,128 to Langer, et al. The more specific use of polymeric delivery systems for administration of antibiotics to bone is described in U.S. Ser. No. 07/810,324 entitled "Bioerodible Polymers for Drug Delivery in Bone" filed Dec. 19, 1991 by Gerhart, et al., the more specific delivery of osteogenic factors is described in U.S. Ser. No. 07/313,953 entitled "Delivery System for Controlled Release of Bioactive Factors" filed February 1989 by Laurencin, et al.
It would be advantageous if muscle morphogenic factors could be identified and delivered to the area around bone or in injured areas, especially if the factors are water soluble and likely to disperse rapidly in vivo, which would be useful in enhancing healing of muscle injuries without a concomitant increase in scarring.
It is therefore an object of the present invention to provide a myogenic differentiating composition.
It is a further object of the present invention to provide a method of restoring functional mammalian muscle with minimal scar formation.
It is another object of the present invention to provide a delivery system for a myogenic differentiating composition.